Investigation of Mutant Hepatitis B Virus in Core Antibody Seropositive Cases of Blood Donor Population
This study was carried out to determine the prevalence
of isolated anti-HBc among blood donors in this province and its impact
on rejection of collected blood units. Isolated hepatitis B core positivity
was found 15% in blood center but in this population we have found no
HBV-DNA positivity. We proposed that in order to detect mutant hepatitis
B viruses in blood donor population, multi-center studies must be done
in this country.
Successes in preventing transmission of viral infections during the last
10 to 20 years have led to very low incidence rates and estimated residual
risk for transfusion-transmitted viral infections (Dodd et al.,
2002). This reduction was primarily achieved by a careful medical selection
of the donors improved sensitivity of serological tests and the introduction
of NAT in minipools for HCV and HIV (Roth et al., 2002a; Eiras
et al., 2003; Stolz et al., 2003). In many studies pooled
or single sample NAT for HBV is advised to confirm safe blood transfusion
in high prevalence areas (Kuhns and Busch, 2006; Matsumoto et al.,
1997; Kleinman et al., 2005).
Enzyme linked immunosorbent assay is the most preferred method in detecting
hepatitis B surface antigen (HBsAg). This method is based mainly on capture
of antigens by antibodies attached on solid phase and recapture them by
using signalled antibodies which detected by an enzymatical reaction (Hoofnagle
and Di Besceglie, 1991; Hoofnagle, 1990).
Post-transfusion hepatitis B is still a relevant subject in spite of
high performance of immunoassays using in hepatitis virus screening (Kojima
et al., 1991).
False sero-negativity has three common causes in traditional methods.
First of all, HBsAg titers may below the range in chronic carrier status.
AntiHBc may be the only marker detected in low level carriers (Jilg et
al., 1995). Second reason is mis-detection of antigenic arrangement
of variant virus type by antibodies on solid phase of assay. In various
geografic regions, vaccine escape mutants may be selected under pressure
of active immunisation and found as dominant strains by means of national
vaccination programs. Detection of vaccine escape mutant strain is more
difficult than wild type virus by traditional methods (Howard and Allison,
1995). Finally, decreased production of hepatitis B surface antigen in
variant strains may cause false sero-negativity (Carman and Mimms, 1997;
Smith and Wu, 2002).
Hepatitis B virus is a common cause of viral hepatitis in worldwide.
More sensitive assays has been developed for screening of blood donors
to prevent transfusion associated hepatitis. Anti-HBc tests are being
used by some countries to detect low level viremia in chronic carriers.
Because of low level viral load in chronic isolated anti-HBc carriers,
PCR tests from plasma pools may give negative results. Screening for anti-HBc
may decrease this risk (Jilg et al., 1995). Seropositivity of anti-HBc
is the major cause of rejection for blood donation. Testing for HBsAg
alone is not fully protective and anti-HBc remains necessary as a screening
test. The presence of anti-HBs is not always indicative of absence of
the replicative virus.
The major aim of this study is to find frequency of Hepatitis B virus
mutants in special blood donor groups (HBsAg weak positive, isolated anti-HBc)
and its importance in rejection of blood donation.
MATERIALS AND METHODS
Blood samples from 174 blood donors were collected between October 2005
and October 2006 in Çanakkale State Hospital Central Laboratory
Unit. All samples were stored at -10°C until the study procedure.
This samples were tested for HBsAg, anti-Hbc IgM and anti-HBs by using
Beckman Coulter (USA) Access 2 immunanalysis system. The study designed
in 3 groups:
||Low level HbsAg positivity (below 5 IU mL-1)
(N = 53)
||Isolated anti-HBc positivity without anti-HBs seropositivity (N
||Anti-HBc positivity with low level anti-HBs seropositivity (below
10 IU mL) (N = 22)
We proposed to investigate mutant hepatitis B virus and false negative
HbsAg test results in our blood donor population. In order to detect false
negative results in our laboratory, control group is selected from serum
samples of 180 HBsAg positive cases from outpatient clinics of Canakkale
State Hospital with cutt-off levels higher than 100 IU mL-1.
Serum specimens stored at -20°C and later studied by using Abbott
Murex, AxSYM V2.
The AxSYM HBsAg V2 assay is based on microparticle immunoassay technology.
Briefly, the sample (150 μL), anti HBs (one monoclonal antibody)-coated
microparticles and biotinylated anti-HBs (poly-clonal antibody) are combined
and incubated in one reaction vessel. Because of poly-clonal antibody
detection, this assay was found most sensitive in recognisation of mutant
HBV antigenic series by many authors (Weber et al., 2003; Taylor
et al., 2004; Thoai et al., 2006).
Finally a pooled sera sample prepared by taking 20 μL serum from
each blood samples, stored at -20°C and later studied PCR by using
ABI PRISM® 5700 DNA sequencer, TaqMan® 1000 RXN PCR Core Reagents.
DNA extraction was achieved by using Nucleospine DNA isolation kit. The
cantitation range of HBV-DNA was accepted as 3x102-3x108
copy mL-1 sera.
RESULTS AND DISCUSSION
There have been found no HBsAg seropositivity by using Abbott Murex,
AxSYM V2 in totally 174 blood samples although all 180 control sera samples
were positive for HbsAg. There is no false negative results and no difference
found in three study group.
After than, pooled sera sample tested with HBV DNA real-time polymerase
chain reaction (ABI PRISM® 5700 DNA sequencer, TaqMan® 1000 RXN
PCR Core Reagents). Pooled sera was found negative for HBV.
We determined the isolated core seropositivity as 15% (179/1196) in our
blood center for study interval period.
The replication of HBV is ongoing in a substantial proportion of healthy
blood donors who have anti-HBc. Blood from such donors may contain very
low levels of HBV free of immune complex formation and should be excluded
for transfusion (Jongerius et al., 1998; Levicnic-Stezinar, 2004).
Collectively, around 30 to 35% of HBsAg-negative subjects with chronic
hepatitis with or HBsAg mutant investigation should be considered when
unusual serologic profiles occur, e.g., for (i) individuals with isolated
anti-HBc reactivity, (ii) patients with discordant results between HBsAg
assays, (iii) patients seronegative for HBsAg but positive for HBeAg and
(iv) individuals with the presence of both HBsAg and anti-HBs (mostly
at low titers of 100 mIU mL-1) (Alhababi et al., 2003).
A study from Canada reports that 3.25% of anti-HBc positive samples (38/1169)
were found to be positive for the presence of HBV DNA in blood (Chevrier
et al., 2007).
In another study from Canada, the proportion of potentially infectious
donations intercepted by anti-HBc screening has been found 1 in 17,800
cases (O`Brein et al., 2007).
In a study from Europe, isolated anti-HBc reactive 104 patient was comparatively
investigated by Elecsys HBsAg and Murex HBsAg assays. Only 1 sample found
(0.96%) to be repetitively reactive by the Murex HbsAg, suggesting that
a mutant form of HbsAg was responsible for the isolated anti-HBc reactivity,
however neutralisation assay was not interpretable and HBV DNA PCR was
negative (Weber et al., 2001). In another study from Germany, after
screening 3.6 million donor samples, 6 HBV PCR-positive, HbsAg-negative
donations were identified (0.0016) and 1 of them was found chronic
anti-HBc positive low-level HBV carrier. Authors of this study states
that, minipool PCR was sensitive enough to identify HbsAg-negative occult
HBV infection in blood donor population (Roth et al., 2002b).
Another study from Pakistan states that isolated anti-HBc reactivity
in HbsAg-negative blood donors is 17.28% (167/966) and of them 2.99% (5/167)
has detectable HBV-DNA which presumptive of occult HBV infection (Bhatti
et al., 2007).
In a large study from Japan, 308 samples from 16 million blood donors
(0.01) which HBsAg negative has been found anti-HBc positive (Tomono
et al., 2002).
Turkey is a medium endemic area about hepatitis B prevalence found 2-7%
in many studies (Balik, 1994). Studies in our country showed that HBV-DNA
positivity rates is 11.3-41% in HbsAg positive cases. But in this studies
authors found no HBV-DNA positivity in HBsAg negative cases (Sahin et
al., 2001; Heper et al., 1999; Ozbilge et al., 2005).
In another study from Turkey, no HBV-DNA positivity was found in isolated
anti-HBc seropositive chronic hepatitis cases (Sonsuz et al., 1992).
We have found no HBV DNA showing that there is no mutant hepatitis B
in this serum samples studied.
And also found that there is 15% isolated HBV core antibody positivity
in our blood center, showing high seroprevalance of hepatitis B. Although
there is no transfusion transmitted acute hepatitis B case for the last
ten years in our province (Health Statistics for Basic Health Services,
Turkish Ministry of Health). This finding may be explained by strict donor
selection by blood centers and high serum antibody titers found in blood
donor population in our province.
Some studies proved that in HBeAg negative subjects, there is a strong
correlation between the serum HBV-DNA and alanine aminotransferase (ALT)
levels; ALT level is usually normal if the samples tested showed an HBV-DNA
level less than 10(5) mL-1 and monitoring of ALT is of value
in assessing hepatocellular damage in patients with chronic hepatitis
B virus infection (Sakugawa et al., 2001; Yalcin et al.,
Many researchers states that isolated core-positive donors may potentially
infectious for HBV so recommends to exclude core-positive donations. However
in countries with high hepatitis B seroprevalance, exclusion of isolated
core antibody positive donors may result difficulty in donation programs.
But for making donations safer this is obviously logical decision. The
anti-HBc test is not obligatory in Turkish Blood Banks controlled by Health
In conclusion we advice screening for HBsAg and anti-HBc with transaminases
to make safer donations. The PCR technology has been used as a routine
clinical test in Turkey for only last 10 years. Nucleic acid assays is
not cost beneficial because of low detection rate in isolated core positive
cases. Clinicians should be educated by transfusion safety programs, because
there is no guarantee for safe blood transfusion especially in high seroprevalance
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